Neurofibromatosis Type 1 is a common genetic disorder in humans, occurring in 1 in 2,500-3,500 live births. Neurofibromatosis Type 1 is caused by inherited or de novo mutation in the Neurofibromin 1 (NF1) tumor suppressor gene, which encodes a GTPase activating protein (GAP) for Ras (rat sarcoma viral oncogene homolog) signaling proteins. Neurofibromatosis Type 1 has a broad clinical spectrum, wherein affected individuals can develop benign nervous system tumors called neurofibromas, low-grade astrocytomas, pheochromocytoma, and juvenile myelomonocytic leukemia (Korf (2000) Oncologist 5:477-85). Plexiform neurofibromas occurring in deep nerves can degenerate into malignant peripheral nerve sheath tumors (MPNST), a life-threatening consequence of Neurofibromatosis Type 1 (Carroll & Ratner (2008) Glia 56:1589-605; Ferner & Gutmann (2002) Cancer Res. 62:1573-7). The lifetime risk of MPNST in Neurofibromatosis Type 1 patients is estimated to be 8% to 15%, and the 5-year survival is approximately 20% (Evans, et al. (2002) J. Med. Genet. 39:311-4; McGaughran, et al. (1999) J. Med. Genet. 36:197-203; Porter, et al. (2009) Sarcoma 2009:756395).
Plexiform neurofibromas are heterogeneous, composed of fibroblasts, perineurial cells, mast cells, and Schwann cells, but only Schwann cells have biallelic inactivation of NF1 (Rutkowski, et al. (2000) Hum. Mol. Genet. 9:1059-66). In mouse models, targeted deletion of NF1 from the Schwann cell lineage gives rise to neurofibromas (Zhu, et al. (2002) Science 296:920-2; Wu, et al. (2008) Cancer Cell 13:105-16; Zheng, et al. (2008) Cancer Cell 13:117-28). Thus, loss of NF1 from Schwann cell precursors is thought to initiate plexiform neurofibroma. Aberrant signaling occurs between NF1-deficient Schwann cells and NF1 heterozygous mast cells, which generates a tumorigenic microenvironment (Zhu, et al. (2002) Science 296:920-2; Yang, et al. (2008) Cell 135:437-48; Monk, et al. (2007) Neuron Glia Biol. 3:233-44). Because of their role in the initiation of plexiform neurofibroma and progression to MPNST, NF1-deficient Schwann cells represent an ideal population for targeted molecular therapies.
Chemical screens have revolutionized the discovery process for targeted molecular therapies. However, primary Schwann cells are difficult to culture and present a challenge for high-throughput screening. Another challenge in drug discovery is the rapid and efficient identification of the receptor for a novel compound—either the physical ligand or the biological process that is being modified. Approaches addressing these challenges are needed to identify new compounds and target pathways for the devastating tumors that afflict Neurofibromatosis Type 1 patients.
The budding yeast Saccharomyces cerevisiae has two NF1 homologues, IRA1 and IRA2, which encode Ras-GAPs (Tanaka, et al. (1990) Cell 60:803-7). Deletion of an IRA gene increases Ras-GTP and activates two pathways, a mitogen-activated protein kinase pathway that modifies cell morphology and the cyclic AMP dependent protein kinase (PKA) pathway (Mosch, et al. (1996) Proc. Natl. Acad. Sci. USA 93:5352-6; Toda, et al. (1985) Cell 40:27-36). Schwann cells lacking NF1 have increased intracellular cyclic AMP and display PKA-dependent phenotypes (Kim, et al. (2001) J. Neurosci. 21:1110-6; Xu, et al. (2002) J. Neurosci. 22:9194-202). The fact that Schwann cells lacking NF1 and budding yeast lacking IRA2 share the high PKA phenotype indicates that the yeast model is useful for targeting the cell-autonomous effects of NF1 loss in Schwann cells. The yeast platform enables rapid and cost effective high-throughput chemical screening and allows for the use of powerful yeast genetics to identify new drug targets.
In this respect, high-throughput chemical screens in mammalian MPNST cell lines and in yeast have been carried out to identify therapeutic agents and target pathways for Neurofibromatosis Type 1-associated tumors. See Wood, et al. (2011) Mol. Cancer Ther. 10:1740, US 2012/0302581, US 2013/0345268, and US 2010/0209931.
Isoxazoloanthrones are a class of compounds which have been described for use in treating HCV infection (see US 2005/09143433) and for inhibiting Jun N-terminal Kinase (JNK) and treating or preventing a disease associated with modulation of JNK (see U.S. Pat. No. 7,354,947).